Species Identification of Silks from Bombyx mori, Eri Silkworm and Chestnut Silkworm Using Western Blot and Proteomics Analyses

Abstract

Species identification is of key significance for exploring the origin and transmission of ancient silks. In this study, two novel methods, i.e. western blot (WB) and proteomics analyses, were proposed and established to identify the differences between silks from Bombyx mori (B. mori) and two other distinctive species (Eri silkworm and Chestnut silkworm). Three diagnostic antibodies, a polyclonal anti-silk fibroin (anti-SF) antibody (pAb), a polyclonal anti-SF-specific peptide antibody (pAsb), and a monoclonal anti-SF antibody (mAb) were designed and prepared to distinguish silk species using the antibody-based WB technique. Proteomics analysis by liquid chromatography-tandem mass spectrometry was performed to further identify silk species at the protein level. WB results indicated that the three antibodies showed high specificity and affinity and could discern B. mori silk from Eri and Chestnut silks. Biomarkers for each SF were obtained using proteomics analysis, and they have the potential to serve as standards for identifying silk species. Thus, combining WB and proteomics analyses with conventional methods can provide more accurate silk information and may be suitable for identifying other proteinaceous materials in archaeological field.

Matrix-assisted laser desorption/ionization time-of-flight mass spectrometry-based biotyping of silk. I. Method development

“…For detailed proteomic analysis of silk, several solvents must be used for the solubilization of fibroin prior to analysis [12][13][14] . Approximately 1 week was required for solubilization, including dialysis, 1 d for trypsinization, and 1 h for MS analysis.…”

“…As sericin was soluble in heated alkaline conditions, stacked bands over 97 kDa were considered sericin (Fig. 1A) -sericin and fibroin are difficult to separate by conventional electrophoresis without intense pre-treatments using several solvents for solubilization [12][13][14] .…”

“…Unfortunately, except for sericin, detailed biochemical analyses of silk proteins have been insufficient owing to their insolubilities 11) . However, recently, proteomic analyses of silk proteins were repeatedly reported following multi-step pre-treatments for solubilization of silk using several solvents [12][13][14] .…”

Degradation Behavior and Immunological Detection of Silk Fibroin Exposure to Enzymes

“…The protocols of producing the primary antibody were narrated in our previous report. 16 The concentration of mAb-BM was 1.0 mg/mL.…”

Matrix-assisted laser desorption/ionization time-of-flight mass spectrometry-based biotyping of silk. II. Differences in 34 local silkworm race cocoons

“…Except for sericin, however, detailed biochemical analyses of silk proteins have not been performed because they are insoluble and difficult to analyze using proteolytic enzymes 11) . Although proteomic analyses of silk proteins became possible after solubilization using several solvents, it is expensive and time consuming [12][13][14] . To solubilize the silk proteins, approximately 1 week was required to complete the procedure.…”